This invention relates generally to epitope-tagged proteins and to transgenic animals expressing such proteins. More specifically, this invention relates to epitope-tagged prion protein (PrP) genes, transgenic animals expressing epitope-tagged PrP genes, and assay.methods for distinguishing between and isolating infectious and noninfectious prion proteins.
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (xe2x80x9cPrPxe2x80x9d) (Bolton et al. (1982) Science 218:1309-11; Prusiner et al. (1982) Biochemistry 21:6942-50; McKinley et al. (1983) Cell 35:57-62). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPC is encoded by a single-copy host gene (Basler et al. (1986) Cell 46:417-28) and is normally found at the outer surface of neurons. A leading hypothesis is that prion diseases result from conversion of PrPC into a modified scrapie isoform called PrPSc during a post-translational process (Borchelt et al. (1990) J. Cell Biol. 110:743-752). It is likely that a fundamental event in the propagation of prions is the conformational transition of alpha-helices in PrPC into beta-sheets in PrPSc (Pan et al. (1993) Proc. Natl. Acad. Sci. 90:10962-10966). Genetic evidence from transgenic mouse studies demonstrates the requirement for an additional component(s) referred to as protein X in this conversion (Telling et al. (1995) Cell 83:79-90).
It appears that PrPSc is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans (see, Prusiner (1991) Science 252:1515-1522). The most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith and Wells (1991) Microbiol. Immunol. 172:21-38). Four prion diseases of humans have.been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) (Gajdusek (1977) Science 197:943-960; Medori et al. (1992) N. Engl. J. Med. 326:444-449). The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Most CJD cases are sporadic, but about 10-15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene (Hsiao et al. (1990) Neurology 40:1820-1827; Goldfarb et al. (1992) Science 258:806-808); Kitamoto et al. (1994) Proc. R. Soc. Lond. 343:391-398). Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts (Brown et al. (1992) Lancet 340:24-27). attempts to link CJD to an infectious source such as the consumption of scrapie infected sheep meat, none has been identified to date (Harries-Jones et al. (1988) J. Neurol. Neurosurg. Psychiatry 51:1113-1119) except in cases of iatrogenically induced disease. On the other hand, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism (Alpers (1979) Slow Transmissible Diseases of the Nervous System, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90).
The initial transmission of CJD to experimental primates has a rich history beginning with William Hadlow""s recognition of the similarity between kuru and scrapie. In 1959, Hadlow suggested that extracts prepared from patients dying of kuru be inoculated into non-human primates and that the animals be observed for disease that was predicted to occur after a prolonged incubation period (Hadlow (1959) Lancet 2:289-290). Seven years later, Gajdusek, Gibbs and Alpers demonstrated the transmissibility of kuru to chimpanzees after incubation periods ranging from 18 to 21 months (Gajdusek et al. (1966) Nature 209:794-796). The similarity of the neuropathology of kuru with that of CJD (Klatzo et al. (1959) Lab Invest. 8:799-847) prompted similar experiments with chimpanzees and transmissions of disease were reported in 1968 (Gibbs, Jr. et al. (1968) Science 161:388-389). Over the last 25 years, about 300 cases of CJD, kuru and GSS have been transmitted to a variety of apes and monkeys.
The expense, scarcity and often perceived inhumanity of such experiments have restricted this work and thus limited the accumulation of knowledge. While the most reliable transmission data has been said to emanate from studies using non-human primates, some cases of human prion disease have been transmitted to rodents but apparently with less regularity (Gibbs, Jr. et al. (1979) Slow Transmissible Diseases of the Nervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 87-110; Tateishi et al. (1992) Prion Diseases of Humans and Animals, Prusiner et al., eds. (London: Ellis Horwood), pp. 129-134).
The infrequent transmission of human prion disease to rodents has been cited as an example of the xe2x80x9cspecies barrierxe2x80x9d first described by Pattison in his studies of passaging the scrapie agent between sheep and rodents (Pattison (1965) NINDB Monograph 2, D. C. Gajdusek, C. J. Gibbs Jr. and M. P. Alpers, eds. (Washington, D.C.: U.S. Government Printing), pp. 249-257). In those investigations, the initial passage of prions from one species to another was associated with a prolonged incubation time with only a few animals developing illness. Subsequent passage in the same species was characterized by all the animals becoming ill after greatly shortened incubation times.
The molecular basis for the species barrier between Syrian hamster (SHa) and mouse (Mo) was shown to reside in the species-specific differences in the sequence of the PrP (Scott et al. (1989) Cell 59:847-857). Mouse PrP (MoPrP) differs from Syrian hamster PrP (SHaPrP) at 16 positions out of 254 amino acid residues (Basler et al. (1986), Cell supra; Locht et al. (1986) Proc. Natl. Acad. Sci. USA 83:6372-6376). Transgenic mice expressing SHaPrP [Tg(SHaPrP)] had abbreviated incubation times when inoculated with SHa prions. When similar studies were performed with mice expressing the human, or ovine PrP transgenes, the species barrier was not abrogated, i.e., the percentage of animals which became infected were unacceptably low and the incubation times were unacceptably long (Telling et al. (1994) Proc. Natl. Acad. Sci. 91:9936-9940; Telling et al. (1995) Cell 83:79-90). Thus, it was not possible to render non-human animals such as mice, susceptible to infection by human prions.
Purification of PrPSc has been facilitated by its relative resistance to proteolytic degradation and insolubility in non-denaturing detergents (Bolton et al. (1982) supra; Prusiner et al. (1982) supra. Purification of PrPC has been more problematic. Immunoaffinity chromatography purification of PrPC yielded only small amounts of protein. Improved purification of PrPC has been accomplished by a multi-step purification procedure involving detergent extraction and separation by immobilized Cu2+ ion affinity chromatography followed by cation-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Pan et al. (1992) Protein Sci. 1:136-144).
The production of monoclonal antibodies against PrPC and PrPSc has been particularly difficult. In the case of mouse PrP, MoPrP is recognized as self, precluding the production of anti-MoPrP antibodies in animals immunized with MoPrP.
There is an urgent need to develop diagnostics and therapeutics for PrPSc-mediated diseases such as CJD. Although many lines of evidence support the idea that PrPC is converted to the infectious PrPSc isoform, greater understanding of the conditions under which scrapie infectivity is generated de novo is needed to develop compounds able to inhibit the generation of PrPSc. Compounds able to inhibit the in vitro conversion of PrPC to PrPSc could be useful for the treatment and prevention of prion-mediated diseases in animal and human subjects at risk. Improved methods for monitoring the conversion of PrP from the alpha-helical conformation of PrPC to the beta-sheet conformation of the infectious PrPSc isoform would be useful in developing assays for such compounds.
Nucleotides encoding a strong epitope tag are operatively placed in a nucleotide sequence encoding a protein which normally has two or more conformational shapes. Depending on the conformational shape assumed by the expressed protein, the tag will or will not be exposed thereby making it possible to differentiate between conformational shapes via an antibody which binds to the epitope. An aspect of the invention features a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence comprising a biologically active protein or protein fragment connected, preferably directly, to a heterologous epitope domain. The expressed amino acid sequence (i.e., the epitope-tagged protein) preferably retains the biological activity of the corresponding natural (e.g., untagged) protein or protein fragment. The tag may be used in connection with a protein which has two or more different conformational shapes, such that the epitope tag is relatively more exposed in one conformational shape relative to another conformational shape.
One aspect of the invention is a transgenic animal such as a mouse which has incorporated into its genome a first DNA sequence encoding a protein which when expressed assumes two or more different conformational shapes. The first DNA sequence has a second DNA sequence encoding an epitope tag connected to it. The second sequence is preferably positioned relative to the first sequence such that the exposure of the tag after expression changes with the different conformational shapes assumed by the protein expressed by the first sequence. The first DNA sequence is preferably an exogenous sequence which encodes a protein such as PrP which protein causes a disease in one conformational shape but not another. Thus by correctly positioning the second sequence encoding the tag relative to the first sequence, it is possible to quickly and easily assay a sample from the animal and determine which conformation the protein has assumed.
Transgenic mammals comprising a tagged transgene are preferably selected from the group consisting of Mus, Rattus, Oryctolagus and Mesocricetus. Transgenic animals expressing high levels of the tagged transgene may be obtained, for example, by over-expression of the transgene with an enhanced promoter and/or with high copy numbers of the transgene.
In a specific embodiment, the invention features a transgenic mammal having an epitope-tagged PrP gene. The PrP gene may be a natural, synthetic, or chimeric PrP gene. In specific embodiments, the transgenic animals have an epitope-tagged chimeric PrP gene which renders the transgenic animals susceptible to infection with a prion which generally only infects a genetically diverse or distinct animal. A chimeric PrP gene is a gene which includes a portion of a gene of a genetically diverse animal. When the transgenic animal is a one of Mus, Rattus, Oryctolagus, or Mesocricetus, the genetically diverse or distinct animal is selected from the group consisting of Bos, Ovis, Sus, and Homo. A preferred transgenic animal is a mouse expressing a epitope-tagged chimeric PrP in which a segment of mouse (Mo) PrP is replaced with the corresponding human (Hu) PrP sequence.
The transgenic animal may be heterozygous or homozygous for an ablated or disrupted endogenous PrP gene; in a preferred embodiment, the transgenic animal is homozygous for an ablated endogenous PrP gene.
In a preferred embodiment of the invention, the epitope-tagged protein is a natural, synthetic, or chimeric prion protein (PrP). PrP may be tagged with a variety of natural or artificial heterologous epitopes known in the art, including artificial epitopes such as FLAG, Strep, or poly-histidine peptides. FLAG peptides include the sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:1) or Asp-Tyr-Lys-Asp-Glu-Asp-Asp-Lys (SEQ ID NO:2). The Strep epitope has the sequence Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO:3). Another commonly used artificial epitope is a poly-His sequence having six histidine residues (His-His-His-His-His-His) (SEQ ID NO:4). Naturally-occurring epitopes include the influenza virus hemagglutinin sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ile-Glu-Gly-Arg (SEQ ID NO:11) recognized by the monoclonal antibody 12CA5 (Murray et al. (1995) Anal. Biochem. 229:170-179) and the eleven amino acid sequence from human c-myc recognized by the monoclonal antibody 9E10 (Glu-Gln-Lys-Leu-Leu-Ser-Glu-Glu-Asp-Leu-Asn) (SEQ ID NO:12) (Manstein et al. (1995) Gene 162:129-134). Another useful epitope is the tripeptide Glu-Glu-Phe which is recognized by the monoclonal antibody YL 1/2 against xcex1-tubulin. This tripeptide has been used as an affinity tag for the purification of recombinant proteins (Stammers et al. (1991) FEBS Lett. 283:298-302).
In a particularly preferred embodiment, the epitope tagged PrP molecule has an artificial FLAG epitope inserted after codon 22, e.g., the first codon of the FLAG epitope begins at codon 23 of a nucleotide sequence encoding a FLAG-tagged PrP protein. The FLAG-tagged PrP molecule retains all of the biological activity of the natural PrP molecule. Specifically, the FLAG-tagged PrP protein retains the ability to support prion propagation.
The invention further includes cells, e.g., omnipotent and pluripotent cells, and immortalized cell lines expressing the epitope-tagged protein construct, as well as transgenic animals having a gene encoding an epitope-tagged protein integrated into their genome.
In another aspect, the invention features a method for distinguishing between the conformational shapes of a protein having a first and second conformation shape, comprising the steps of: (a) generating a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence comprising a protein fragment tagged with a heterologous epitope; b) transfecting a cell or organism with the tagged protein construct; c) expressing the tagged protein. The epitope tag is positioned relative to the protein sequence such that the epitope is exposed on the surface of the tagged protein to a greater degree when the protein is in a first conformational shape relative to the degree of exposure of the epitope when the protein is in a second conformational shape. In one embodiment, the conformational shapes of the protein can be distinguished by detecting the presence or absence of the epitope. Multiple different tags can be used if the protein assumes multiple conformations, making it possible to distinguish the conformations via detection of the presence or absence of a series of tags. In another embodiment, the conformational shapes of a protein are distinguished by relatively greater exposure of the epitope tag in one conformational shape than in other conformational shapes. Preferably, the exposure of an epitope tag is 20-100% greater in one conformational shape relative to the second conformational shape; more preferably, the relative exposure is 50-100% greater; most preferably, the relative exposure is 75-100% greater.
In one embodiment, the epitope-tagged protein is PrP, and the epitope tag is placed such that it is unexposed on the surface of the expressed prion protein when it is in the noninfectious alpha-helical PrPC isoform, but the epitope tag is exposed on the surface of the infectious beta-sheet PrPSc isoform.
In another aspect, the invention features a method of isolating PrP by a) generating a recombinant nucleic acid construct comprising a nucleic acid sequence encoding a prion protein having a heterologous epitope domain; b) transfecting a cell or organism with the tagged PrP construct; c) expressing the construct to produce epitope-tagged PrP, where the epitope tag is placed such that it is exposed on the surface of the desired PrP isoform; and d) purifying PrP by immunoaffinity chromatography using an anti-epitope tag antibody. In a specific embodiment, the method of isolating PrP includes an additional step of enriching for PrP prior to purification. This method can be used to isolate, separate and identify either PrPC and/or PrPSc.
In another aspect, the invention features an assay method for detecting infectious prions by a) generating a transgenic animal comprised of an epitope-tagged PrP gene where the epitope tag is relatively more exposed on the surface of the expressed PrP molecule when the molecule has the PrPSc conformation than when the molecule is in the PrPC conformation; b) inoculating the transgenic animal with material suspected of containing infectious prions; and c) detecting the increased presence of epitope-tagged PrP. Detection of increased levels of epitope-tagged PrP results from increased levels of PrP in the infectious PrPSc conformation, thus indicating the presence of infectious PrP particles in the inoculating material. In one preferred embodiment, the transgenic animal expresses a bovine-mouse MBov2M chimeric PrP gene and is inoculated with material from infected cattle. In another preferred embodiment, the transgenic animals expresses a chimeric human-mouse MHu2M PrP molecule and is inoculated from material from an infected human.
One object of the invention is to provide a transgenic animal producing large quantities of an epitope-tagged protein or protein fragment which is easily purified via immunoaffinity chromatography using an epitope-specific antibody. This is particularly useful where the protein is difficult to purify in sufficient quantities and/or attempts to produce antibodies specific to the protein and its conformation isoforms have been unsuccessful, e.g., PrPC and PrPSc. Additionally, the invention allows a simplified one-step enrichment of PrPC and/or PrPSc, which can be followed by a variety of procedures including immunodetection.
Another object is to provide a transgenic animal expressing elevated levels of a tagged protein or protein fragment obtained with an enhanced promoter or a high copy number of a tagged transgene.
Another object is to provide a method for distinguishing conformational changes in a protein, e.g., distinguishing between the isoforms of PrPC and PrPSc.
Another object is to provide a gene tagged with a heterologous epitope.
Another object of the invention is to provide a transgenic host mammal (which is small, e.g., less than 1 kg when full grown, and inexpensive to maintain) such as a mouse, rat or hamster which includes an exogenous or chimeric PrP gene, including all or a portion of a PrP gene from another animal, (which is large, greater than 2 kg when full grown, and expensive to maintain) such as a human, cow, pig, sheep, cat or dog, and having a artificial epitope tag domain.
Another object of the invention is to provide a transgenic host animal which includes elevated levels of expression of a tagged PrP gene of a genetically diverse animal wherein the elevated levels of expression are obtained by the inclusion of a high copy number of the tagged PrP gene of the genetically diverse mammal and/or fusing an enhanced promoter to the PrP gene of the genetically diverse animal.
One advantage of the method of the invention is the production of elevated levels of readily isolatable PrPC and PrPSc.
Another object is to provide a transgenic animal assay which animal, on inoculation, develops PrPSc which is detectable via an epitope tag as distinguished from PrPC.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the compositions, composition components, methods and method steps of the invention as set forth below.